The single‐atom enzyme (SAE) is a novel type of nanozyme that exhibits extraordinary catalytic activity. Here, we constructed a PEGylated manganese‐based SAE (Mn/PSAE) by coordination of single‐atom manganese to nitrogen atoms in hollow zeolitic imidazolate frameworks. Mn/PSAE catalyzes the conversion of cellular H2O2 to .OH through a Fenton‐like reaction; it also promotes the decomposition of H2O2 to O2 and continuously catalyzes the conversion of O2 to cytotoxic .O2− via oxidase‐like activity. The catalytic activity of Mn/PSAE is more pronounced in the weak acidic tumor environment; therefore, these cascade reactions enable the sufficient generation of reactive oxygen species (ROS) and effectively kill tumor cells. The prominent photothermal conversion property of the amorphous carbon can be utilized for photothermal therapy. Hence, Mn/PSAE exhibits significant therapeutic efficacy through tumor microenvironment stimulated generation of multiple ROS and photothermal activity.
Cysteine proteases are an important class of enzymes involved in the degradative processing of peptides and proteins. 1,2 They are ubiquitous in nature and play vital roles in numerous physiological processes including arthritis, osteoporosis, Alzheimer's disease, cancer cell invasion, and apoptosis. 1-3 Cysteine proteases are also essential to the life cycles of many pathogenic protozoa. 4,5 One such parasite is Trypanosoma cruzi, the etiologic agent of Chagas' disease. Cruzain, 6,7 the major cysteine protease of T. cruzi, has been identified as a potential therapeutic target for treatment of Chagas' disease. [7][8][9] Several strategies have been pursued in the design of cysteine protease inhibitors. 2,10,11 Peptidyl aldehydes, 12 diamino ketones, 13 and nitriles 14 are reversible inhibitors that form hemithioacetals, peptide ketals, and thioimidates, respectively, with the thiol of the active site cysteine residue, mimicking the initial covalent enzyme adduct in normal proteolytic turnover. Epoxysuccinyl derivatives, 15 peptidyl Michael acceptors, 16-18 (acyloxy)methyl ketones, 19 and halomethyl ketones are examples of inhibitors which irreversibly inactivate cysteine proteases via alkylation of the active site cysteine residue. Several classes of nonpeptidic reversible inhibitors of cysteine proteases also have been described. [20][21][22] In connection with efforts to develop potent and selective inhibitors of cruzain, we became interested in the vinyl sulfone inhibitor series first introduced by Hanzlik 17 and further developed by Palmer et al. 18,23 Compound 1a is a potent and selective inhibitor of cruzain, with a second-order rate constant (k inact /K i ) of 203 000 s -1 M -1 . 18 Inhibitors 1a and especially 1b have also proven highly effective against T. cruzi, both in tissue culture and in vivo experiments (mouse model). 24 Although considerable effort has been devoted to the optimization of interactions of inhibitors with the cruzain S 1 and S 2 binding sites, 18,23,25 virtually nothing is known about the interactions of substrates or inhibitors with the S 1 ′ and S 2 ′ sites. The prime site region in cruzain contains a large open surface defined by Trp 177, and available X-ray structures suggest that there is considerable room for prime site inhibitor binding. 7,13,22,25 A recent X-ray structure of cathepsin K, the active site of which is homologous to that of cruzain, with covalently bound APC3328, a dipeptidyl phenyl vinyl sulfone inhibitor related to 1b, reveals that the phenyl residue of the phenyl sulfonyl unit does not make optimal interactions with prime site residues. 26 Accordingly, we decided to probe the possibility that additional selectivity and potency in the vinyl sulfonyl series could be achieved by extending the inhibitor structure into the prime site region, via modification of the sulfonyl substituent as suggested by structure 2. However, we anticipated that it might be easier to synthesize a family of vinyl sulfonamides 3 or vinyl sulfonate esters 4, using the vinyl sulfonyl chl...
The series of vinyl-sulfone-based inhibitors examined in complex with cruzain was designed to probe recognition and binding potential of an aromatic-rich region of the enzyme. Analysis of the interactions formed shows that aromatic interactions play a less significant role, whereas the strength and importance of hydrogen bonding in the conformation adopted by the inhibitor upon binding to the enzyme was highlighted. A derivative of one inhibitor examined is currently under development as a therapeutic agent against Chagas' disease.
[reaction: see text] The relative rates of Michael additions of 2'-(phenethyl)thiol to representative vinyl sulfonyl Michael acceptors were measured. The dependence of the reactivity of the Michael acceptor on the nature of the sulfonyl R substituent was determined in order to evaluate the effect of these substituents on the inactivation kinetics of comparably substituted vinyl sulfonyl cysteine protease inhibitors. The rates of these Michael additions vary over 3 orders of magnitude, with phenyl vinyl sulfonate esters (R = OPh) being ca. 3000-fold more reactive than N-benzyl vinyl sulfonamides (R = NHBn).
to perform PDT. [7] PSs absorb laser energy in the presence of O 2 to produce cytotoxic reactive oxygen species (ROS) such as singlet oxygen ( 1 O 2 ) that causes the destruction of the genetic material in cancer cells, leading to cell apoptosis, or necrosis. [7][8][9][10] The O 2 involved in PDT improves tumor destruction and reduces the toxic side effects as compared with other conventional therapeutic modalities like radiotherapy, chemotherapy, and surgery. [11][12][13][14][15] However, hypoxia, one of the hallmarks of malignant tumors, [16][17][18] induces an unexpected resistance of tumors to PDT, since molecular O 2 plays an essential role during the process. Some types of nanocatalysts have been used to address this dilemma, such as manganese dioxide (MnO 2 ) nanoparticles, carbon dot, and single-atom ruthenium (Ru) for an in situ catalysis of the decomposition of H 2 O 2 to generate O 2 . [6,14,19] This could be an effective strategy to relieve hypoxia in the tumor microenvironment (TME), thus becoming a potential approach to improve the efficacy of PDT. [20] Additionally, the acidic TME with an excessive amount of H 2 O 2 is a natural activator of these nanocatalysts, making them intelligent nanocatalysts for tumor specific therapy. [21][22][23] Recently, MnO 2 nanostructures have received extensive attention in the field of bio-applications for their efficient O 2 production and easy synthesis, [24][25][26][27] enhancing the effect of radiation therapy, [27] chemotherapy, [28] and PDT. [29] In addition, MnO 2 is rapidly decomposed into water soluble Mn 2+ ion in an acidic condition, [6,[30][31][32][33][34] and excreted through the bile into the feces, avoiding unexpected accumulation and long-term toxicity in vivo. [6,29] However, MnO 2 nanostructures without surface coating have a poor structure stability under physiological conditions, [35] and it is difficult to control their size and morphology during the synthesis, thus, increasing the uncertainty of the reactivity of the nanomaterial. [25] Therefore, it is highly desirable to construct MnO 2 nanoparticles with uniform morphology, high stability and biocompatibility for biomedical applications.Ferritin (Ftn) is an endogenous iron storage protein composed of 24 subunits, with a hollow structure of 12 nm in the external diameter and an inner cavity of 8 nm. [36] Ftn has been widely used as a superior protein nanocage for the Hypoxia is a hallmark of the tumor microenvironment (TME) that promotes tumor development and metastasis. Photodynamic therapy (PDT) is a promising strategy in the treatment of tumors, but it is limited by the lack of oxygen in TME. In this work, an O 2 self-supply PDT system is constructed by co-encapsulation of chlorin e6 (Ce6) and a MnO 2 core in an engineered ferritin (Ftn), generating a nanozyme promoted PDT nanoformula (Ce6/ Ftn@MnO 2 ) for tumor therapy. Ce6/Ftn@MnO 2 exhibits a uniform small size (15.5 nm) and high stability due to the inherent structure of Ftn. The fluorescence imaging and immunofluorescence analysis dem...
Background Microglial activation-mediated neuroinflammation plays an important role in the progression of neurodegenerative diseases. Inflammatory activation of microglial cells is often accompanied by a metabolic switch from oxidative phosphorylation to aerobic glycolysis. However, the roles and molecular mechanisms of glycolysis in microglial activation and neuroinflammation are not yet fully understood. Methods The anti-inflammatory effects and its underlying mechanisms of glycolytic inhibition in vitro were examined in lipopolysaccharide (LPS) activated BV-2 microglial cells or primary microglial cells by enzyme-linked immunosorbent assay (ELISA), quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, immunoprecipitation, flow cytometry, and nuclear factor kappa B (NF-κB) luciferase reporter assays. The anti-inflammatory and neuroprotective effects of glycolytic inhibitor, 2-deoxoy-d-glucose (2-DG) in vivo were measured in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-or LPS-induced Parkinson’s disease (PD) models by immunofluorescence staining, behavior tests, and Western blot analysis. Results We found that LPS rapidly increased glycolysis in microglial cells, and glycolysis inhibitors (2-DG and 3-bromopyruvic acid (3-BPA)), siRNA glucose transporter type 1 (Glut-1), and siRNA hexokinase (HK) 2 abolished LPS-induced microglial cell activation. Mechanistic studies demonstrated that glycolysis inhibitors significantly inhibited LPS-induced phosphorylation of mechanistic target of rapamycin (mTOR), an inhibitor of nuclear factor-kappa B kinase subunit beta (IKKβ), and NF-kappa-B inhibitor alpha (IκB-α), degradation of IκBα, nuclear translocation of p65 subunit of NF-κB, and NF-κB transcriptional activity. In addition, 2-DG significantly inhibited LPS-induced acetylation of p65/RelA on lysine 310, which is mediated by NAD-dependent protein deacetylase sirtuin-1 (SIRT1) and is critical for NF-κB activation. A coculture study revealed that 2-DG reduced the cytotoxicity of activated microglia toward MES23.5 dopaminergic neuron cells with no direct protective effect. In an LPS-induced PD model, 2-DG significantly ameliorated neuroinflammation and subsequent tyrosine hydroxylase (TH)-positive cell loss. Furthermore, 2-DG also reduced dopaminergic cell death and microglial activation in the MPTP-induced PD model. Conclusions Collectively, our results suggest that glycolysis is actively involved in microglial activation. Inhibition of glycolysis can ameliorate microglial activation-related neuroinflammatory diseases.
The combination of photodynamic therapy (PDT) and enzyme therapy is a highly desirable approach in malignant tumor therapies as it takes advantage of the spatial-controlled PDT and the effective enzyme-catalyzed bioreactions. However, it is a challenge to co-encapsulate hydrophilic enzymes and hydrophobic photosensitizers, and these two agents often interfere with each other. In this work, a protocell-like nanoreactor (GOx-MSN@MnPc-LP) has been designed for synergistic starvation therapy and PDT. In this nanoreactor, the hydrophilic glucose oxidase (GOx) is loaded in the pore of mesoporous silica nanoparticles (MSNs), while the hydrophobic manganese phthaleincyanide (MnPc) is loaded in the membrane layer of liposome. This spatial separation of two payloads protects GOx and MnPc from the cellular environment and avoids interference with each other. GOx catalyzes the oxidation of glucose, which generates hydrogen peroxide and gluconic acid, leading to the starvation therapy via glucose consumption in cancer cells, as well as the disruption of cellular redox balance. MnPc produces cytotoxic singlet oxygen under 730 nm laser irradiation, achieving PDT. The antitumor effects of the nanoreactor have been verified on tumor cells and tumor-bearing mice models. GOx-MSN@MnPc-LP efficiently inhibits tumor growth in vivo with a single treatment, indicating the robust synergy of starvation therapy and PDT treatment. This work also offers a versatile strategy for delivering hydrophilic enzymes and hydrophobic photosensitizers using a protocell-like nanoreactor for effective cancer treatment.
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